• Implement a two-step endogenous plasmid curing system based on CRISPR-Cas9 method developed by Kan et al. (2020) to reduce EcN metabolic burden


• Assemble the LIRA biodevices generated by the dry lab team into endogenous plasmid backbone (pMUT1) using Gibson Assembly


• Assembly transformation into competent, plasmid-free, EcN cells


• Test for stability and growth of the transformed bacteria

Day 1

1. Prepare overnight Escherichia coli culture on 5 mL of LB broth inside of 15 mL sterile falcon tube from glycerol stocks or plate colonies.



Day 2

2. Inoculate 1% of the overnight pre-culture on to 100 mL LB medium, seal with sterile cotton, alumunium foil, and wrap.


3. Incubate in 37°C for 2-3 hours on 250 rpm up until the OD6000 reaches ~0,3-0,4.


4. Put culture on ice bath for 10 minutes (from this point keep in mind to make sure the Escherichia coli stays cold).


5. Chill microtips, microtubes,cryotubes, and 50 mL falcon tubes before use


6. Transfer equal parts of the culture to 2 50 mL chilled falcon tubes


7. Centrifuge on 2700 rcf for 10 minutes on 4°C


8. Discard medium, resuspent the cell pellet on 1,6 mL of chilled 100 mM MgCl-CaCl on ice


9. Incubate on ice for 30 minutes


10. Centrifuge on 2700 rcf for 10 minutes on 4°C


11. Discard medium, resuspent the cell pellet on 1,6 mL of chilled 100 mM CaCl on ice


12. Incubate on ice for 20 minutes


13. Mix cells into one conical flask, then aff 0,5 mL of chilled 80% glycerol, mix on ice by slowly turning the mixture


14. Transfer 100 μL of the mixture into cryotubes, then freeze with liquid nitrogen


15. Store competent cells on -80°C

1. Thaw competent cells on ice for 10-20 minutes


2. Add 2 μL of plasmid and 50 μL of thawed competent cells into 1,5 mL sterile microtubes


3. Mix by pipetting or flicking the tubes 3-4 times (do not vortex)


4. Put the mixture on ice for 30 minutes


5. Heat shock on 42°C for 45 seconds


6. Transfer the shocked tubes on ice for 5 minutes


7. Add 950 μL of room temp. SOC medium/LB broth onto the microtubes


8. Incubate on 37°C 250 rpm shaker for 60 minutes


9. Warm selection plates on 37°C


10. Spread 100 μL of bacteria into selection plate using disposable spreader/ sterilized glass spreader


11. Centrifuge the remaining 850 μL on 6800 rcf for 3 minutes


12. Discard 700 μL of the supernantant and mix the remaining 100 μL with cell pellet


13. Incubate overnight (14-20h) on 37°C

Day 1

1. Transform pFREE plasmid (for pMUT1 curing) or pCryptdel4.8 (for pMUT2 curing)


2. Incubate overnight (14-20h) on 37°C



Day 2

3. Culture one of the colony into 10 mL LB with added 10 μL of 50 mg/mL kanamycin on 15 mL falcon tubes


4. Incubate overnight (14-20h) on 37°C, static condition



Day 3

5. Culture 10 μL of the preculture into 10 mL 10 mL LB with added 10 μL of 50 mg/mL kanamycin, 100 μL of 20% ramnose stock solution ( 0,2% ramnose), and 0,2 uL of stock anhydrotetracycline (ATC) solution 10 mg/mL (ATC 0,43 uM/0,2 ug/mL) on 50 mL falcon tubes


6. Incubate overnight (14-20h) on 37°C, static condition



Day 4

7. Perform 8x serial dilution by diluting 1 mL of the culture into 9 mL of LB broth media, mix using vortex


8. Spread 1 mL of the diluent into LB agar plates


9. Incubate overnight (14-20h) on 37°C, static condition



Day 5

10. Perform colony PCR using appropriate primers according to MasterMix protocol.

1. Pick Appropriate colonies from overnight selection plates


2. Prepare 100 μL nuclease-free water on 1,5 mL microtubes


3. Isolate picked colonies using sterile toothpick, submerge the toothpick into NFW-filled microtubes


4. Boil colonies on 80°C for 5 minutes


5. Wait for the mixture to cool down, then centrifuge on 5000 rpm, room temperature for 5 minutes


6. Use the supernatant as DNA template in the PCR reaction


7. PCR Mixing (MyTaq 2x MasterMix)



Component	Volume
Template (colony)	5 uL
Primer F	1 uL
Primer R	1 uL
2x PCR master mix	12.5 uL
Nuclease-free water	5.5 uL
Total	25 uL

8. Run PCR according to manufacturer's protocol for 35 cycles


9. Run electrophoresis for PCR product on 1% agarose gel, 100 V for 30 minutes

1. Transfer up to 100 µl of PCR product (excluding oil) to a microcentrifuge tube (not provided) and add 5 volumes of FADF Buffer, mix well by vortexing.


2. Place a FADF column into a Collection Tube.


3. Transfer the sample mixture to the FADF Column. Centrifuge at 11,000 x g for 30 seconds, then discard the flow-through.


4. Add 750 µl of Wash Buffer (ethanol added) to the FADF Column. Centrifuge at 11,000 x g for 30 seconds, then discard the flow-through.


5. Centrifuge again at full speed (~18,000 x g) for an additional 3 minutes to dry the column matrix. Remove the residual liquid thorougly


6. Place the FADF Column to a new microcentrifuge tube (not provided).


7. Add ≥ 20 µl of Elution Buffer or ddH2O to the membrane center of the FADF Column. Stand the FADF Column for 1 min. make sure that the elution solution is dispensed onto the membrane center and is absorbed completely.


8. Centrifuge at full speed (~18,000 x g) for 1 min to elute the DNA

1. Excise the DNA fragment from the agarose gel using a razor blade, scalpel or other device and transfer it into a 1.5 ml microcentrifuge tube.


2. Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g. for 100 µl (mg) of agarose gel slice add 300 µl of ADB).


3. Incubate at 55 °C2 for a minimum of 10 minutes and then briefly mix the sample by vortexing or inverting. For optimal performance, it is essential that the gel slice is completely dissolved before moving on to step 4. For DNA fragments > 8 kb, following the incubation step, add one additional volume (equal to that of the gel slice) of water to the mixture for better DNA recovery (e.g., 100 µl agarose, 300 µl ADB, and 100 µl water).


4. Transfer the melted agarose solution to a Zymo-Spin™ Column in a Collection Tube.


5. Centrifuge for 1 minute. Discard the flow-through


6. Add 200 µl of DNA Wash Buffer to the column and centrifuge for 30 seconds. Discard the flow-through. Repeat the wash step.


7. Add ≥ 6 µl DNA Elution Buffer4 or water5 directly to the column matrix.


8. Place the column into a 1.5 ml tube and centrifuge for 1 minute to elute DNA. Ultra-pure DNA is now ready for use.

Insert and Gibson primer design

Overhang generation (mutagenesis) using Hi-Fi PCR Mix

1. Dissolve dried DNA according to the manufacturer instructions


2. Dilute to recommended concentration according to PCR protocol


3. Mix PCR reagents into PCR tubes (20 uL reactions)



Component	20 µL rxn	Final conc.
NFW	Add up to 20 µL
5X Phusion™ HF	4 uL	1x
Buffer[1]
10 mM dNTPs	0.4 µL	200 µM each
F primer	X µL	0.5 µM
R primer	X µL	0.5 µM
Template DNA	X µL	1 pg - 10 ng/50 µL
Phusion™ High–Fidelity DNA Polymerase	0.2 µL	0.02 U/µL

4. Run PCR reactions per recommended protocol for 35 cycles, use optimized annealing temperature from previous optimization

Gibson Assembly Reaction

1. Mix the DNA segments and the backbone in one tube. Yield will be the best if each DNA fragment has equimolar concentration.


2. Mix the DNA fragment and add the Gibson Cloning Master Mix.


3. Incubate for 1 hour in 50ºC.


4. Assembly results, along with the positive and negative control were then transformed into competent DH5α provided by the manufacturer and EcN cells


5. Spread transformants into selection plates


6. Incubate overnight at 37ºC

1. Mix the DNA segments and the backbone in one tube. Yield will be the best if each DNA fragment has equimolar concentration.


2. Mix the DNA fragment and add the Gibson Cloning Master Mix.


3. Incubate for 1 hour in 50ºC.


4. Assembly results, along with the positive and negative control were then transformed into competent DH5α provided by the manufacturer and EcN cells


5. Spread transformants into selection plates


6. Incubate overnight at 37ºC


7. Centrifuge 0.5-5 ml1 of bacterial culture in a clear 1.5 ml tube at full speed for 15-20 seconds in a microcentrifuge. Discard supernatant.


8. Add 250 µl of cold ZymoPURE™ P1 (Red) to the bacterial cell pellet and resuspend completely by vortexing or pipetting.


9. Add 250 µl of ZymoPURE™ P2 (Blue) and immediately mix by gently inverting the tube 8-10 times. Do not vortex! Let sit at room temperature for 3 minutes2. Cells are completely lysed when the solution appears clear, purple, and viscous.


10. Add 250 µl of ZymoPURE™ P3 (Yellow) and mix thoroughly by inversion. Do not vortex! Invert the tube an additional 5 times after the sample turns completely yellow. The sample will turn yellow when the neutralization is complete, and a yellowish precipitate will form.


11. Centrifuge the neutralized lysate for 5 minutes at 16,000 x g.


12. Transfer exactly 600 µl of supernatant from step 5 into a clean 1.5 ml microcentrifuge tube.


13. Add 260 µl of ZymoPURE™ Binding Buffer to the cleared lysate from step 6 and mix thoroughly by vortexing for 15 seconds3.


14. Place a Zymo-Spin™ II-PX Column in a Collection Tube.

1. Transfer the entire mixture from step 7 into the Zymo-Spin™ II-PX Column. Incubate the Zymo-Spin™ II-PX/Collection Tube assembly at room temperature for 1 minute and then centrifuge at ≥ 10,000 x g for 1 min. Discard the flow through


2. Add 800 µl of ZymoPURE™ Wash 1 to the Zymo-Spin™ II-PX Column and centrifuge at ≥ 10,000 x g for 1 min2. Discard the flow through.


3. Add 800 µl of ZymoPURE™ Wash 2 to the Zymo-Spin™ II-PX Column and centrifuge at ≥ 10,000 x g for 1 min. Discard the flow through.